1. Field of the Invention
This invention relates to an improved method for the biosynthesis of proteins.
2. Description of the Related Art
There are several methods for synthesizing proteins.
One method is in vivo synthesis. In vivo synthesis has been the primary method for synthesizing proteins because it has produced higher yields than has in vitro synthesis. But a main disadvantage of in vivo synthesis of recombinant proteins is that the proteins produced by in vivo synthesis are often improperly folded.
As suggested in the preceding paragraph, a second major category of synthesizing proteins is in vitro biosynthesis. As used herein, in vitro biosynthesis means cell-free protein synthesis using either mRNA (translation system) or its complementary DNA (coupled transcription/translation system) as the template for protein synthesis, which is added to a cell extract containing the other biological components, e.g., ribosomes, t-RNAs, aminoacyl-tRNA synthetases, nucleotides, amino acids, etc., needed for protein synthesis.
Classical cell-free protein synthesis in a batch mode is, however, inefficient, i.e., it produces a low yield of the desired proteins. And continuous-flow cell-free synthesis has two significant disadvantages. Although continuous-flow cell-free synthesis as proposed by Spirin (Spirin, A. S. et al. (1988), Science 242, 1162-1164) initially produces a good yield, the desired rate of protein production does not continue for a sufficiently long time to be commercially useful, possibly due to the elution of important components, such as proteins and tRNAs, from the reactor through a semipermeable membrane (Endo, Y. et al. (1992), J. Biotech. 25, 221-230). Also, other researchers have determined that the activity of synthesized enzymes produced by this method decreases with the time of the synthesis reaction due to defects in protein folding. Misfolding is thought also, at least in part, to cause clogging of the semipermeable membrane in a continuous system. (Kudlicki, Wieslaw; Kramer, Gisela; and Hardesty, Boyd, "Cell Free System for Protein Synthesis and Use of Chaperone Proteins Therein," International Application Number PCT/US94/03860, International Publication Number WO 94/24303, International Publication Date Oct. 27, 1994 and Nishimura, Norihiro; Kitaoka, Yoshittisa; and Niwano, Mitsuru (1995) "Enhancement of Protein Synthesis in Continuous-Flow, Cell-Free System by Improvement of Membrane Permeation," Journal of Fermentation and Bioengineering, volume 80, number 4, pp. 403-405)
Furthermore, the yield of the protein synthesized by a third distinct technique for synthesizing proteins, i.e., a chemical method, such as the one used by Merrifield and described in Merrifield, B. (1986) Science 232, 341-347, unfortunately decreases with the length of the synthesized protein. This is due to the fact that errors are introduced because the coupling of each amino acid to the partially completed polypeptide chain is not one hundred percent efficient. The population of proteins with such errors, i.e., amino acid deletions, increases with each amino acid addition. For this reason it is not commercially feasible to make large quantities of long polypeptide chains with this method.
To produce a high yield of desired proteins, it would, therefore, be advantageous to have a continuous-flow cell-free technique which produces properly folded proteins.
In a cell, protein folding is assisted by at least two groups of proteins, viz., (1) enzymes (such as protein disulphide isomerase and peptidyl prolyl cis-trans isomerase) which catalyze isomerization of specific peptide bonds and (2) molecular chaperones (e.g., proteins of the hsp70). hsp90, and chaperonin (GroEL/hsp60) families) which prevent inappropriate protein-protein interactions that would, if not prevented, lead to incorrectly folded and, thus, inactive, proteins. However, these naturally existing systems which assist protein folding often fail upon a synthesis of large quantities of recombinant protein, both in cells and in cell-free systems. This, consequently, leads to the production of large quantities of inactive protein.
The present inventors are, furthermore, aware that an affinity matrix has been utilized to isolate a protein during renaturing as part of a rather complicated process. In that process proteins were synthesized in cells. Then the cells were broken. The synthesized eukaryotic proteins were then in an agglomeration. Therefore, such proteins had to be denatured. The affinity tag and the affinity matrix were employed in the subsequent renaturing--for cases when renaturing could, in fact be achieved--to isolate the protein and sometimes achieve proper folding.